The orchestration of cell behavior during complex morphogenesis involves not only the well-studied biochemical pathways but also an important and fascinating biophysical system: the endogenous patterns of ion flows, transmembrane gradients, and electric fields produced by ion channels and pumps. Such bioelectrical signals are produced from all cells (not just nerve and muscle) and regulate cell proliferation, migration, and differentiation. Bioelectrical gradients are crucial determinants of patterning in development, regeneration, and resistance to neoplasm. Our laboratory has pioneered the development of novel techniques for the molecular-level characterization of bioelectrical signals and genetic gain- and loss-of-function approaches for their rational modulation in vivo. Over the last decade, we have uncovered several new roles for transmembrane potential gradients in stem cell regulation, left-right patterning, tail regeneration, eye induction, and planarian anterior-posterir polarity determination. Moreover, we have identified the molecular steps that transduce voltage changes into transcriptional responses, thus fleshing out the mechanistic links between physiological controls of growth and form and downstream genetic components that regulate cell behavior. Strong data now indicate that modulation of bioelectric properties of cells has the potential to revolutionize approaches in regenerative medicine of the face, eye, spinal cord, and limb, as well as detection and normalization of cancer. In this R03 project, we propose to surmount the biggest barrier facing this field today: the difficulty of changing and monitoring transmembrane potential in vivo with very high spatio-temporal resolution. Preliminary data indicate that misexpression of specific ion channels can induce whole, complete, functional forelimbs in frogs; this is a superb opportunity to understand this new type of developmental signaling. What is holding back the community from fully cracking the bioelectric code is a proof-of-concept application that allows spatial patterns of bioelectric gradients to be regulated in viv. Our basic idea is to move the technology of optogenetics, heretofore applied only to excitable cells (nerve and muscle), to all cell types for developmental regulation. We propose to: 1) produce transgenic frogs expressing voltage fluorescent reporter proteins, facilitating the investigation of bioelectrical properties in any organ/tissue of interest in vivo; 2) produce transgenic frogs expressing optogenetic constructs (Channelrhodopsin and Archeorhodopsin), allowing depolarization or hyperpolarization of cells of interest in vivo by means of patterned light exposure; and 3) use this technology to induce ectopic limbs in froglets by developing a mechanistic understanding of the voltage gradients specific to limb induction and morphogenesis. By creating powerful and widely-applicable resources and applying them to a problem of fundamental importance and biomedical relevance (limb damage), our work will provide a key proof-of-principle to stepping stone to advance the field of optogenetics, the regenerative medicine of the vertebrate limb, and the understanding of biophysical factors in development.